The proposed research continues a genetic investigation of mitochonddal inheritance. Mitochondria are universally inherited only from the egg, not from the sperm. Rare instances of abnormal inheritance of paternal mitochondria in humans can result in developmental problems or metabolic disorders. Recent cell biological studies have shown that mammalian sperm mitochondria are marked with ubiquitin, the universal degradation signal in eukaryotic cells. However, we do not yet know the suite of genes involved in this process, and whether this suite includes only sperm genes or both sperm and egg genes. Also, it is not clear whether non-mammalian taxa use ubiquitin for paternal mitochondrial degradation, in our previous studies with the nematode C. elegans, we have generated four mutant strains that fail to eliminate the sperm mitochondria from the embryo. These are the first such mutants ever generated in any species. The research proposed here will continue our genetic investigations of the mechanism of paternal mitochondrial destruction in C. elegans using the powerful genetic and genomic tools available for this species. Specifically, we aim to test the hypothesis that sperm mitochondria are uniquely tagged for destruction inside the embryo. We will continue our random mutagenesis for mutations that block the degradation of sperm mitochondria. Our previous mutations and new ones we isolate will be mapped and their sequence determined. We will test a second hypothesis that the oocyte actively recognizes the sperm mitochondria and degrades them. We will mutegenize and select for mutant oocytes that fail to eliminate the sperm mitochondria, and we will then map and determine the sequence of the mutated genes. Finally, we will test a third hypothesis that ubiquitin is the degradation label for sperm mitochondria. In a collaborative study, we will continue to knock out sperm specific genes that are associated with the ubiquitination pathway and examine the mutants for paternal mitochondrial inheritance. This project represents a comprehensive effort to study mitochondrial inheritance, and it provides the first genetic approach to the problem. Furthermore, identification of the genes involved in paternal mitochondrial degradation will help elucidate the mechanism of mitochondrial inheritance.